Under feeding conditions, elevations in blood glucose and circulating incretin hormones such as glucagon like peptide-1 (GLP-1) stimulate islet survival in part via the activation of the transcription factor CREB (Jhala et al., 2003). Elevations in glucose stimulate insulin secretion and islet cell gene expression via closure of KATP channels and subsequent influx of calcium through activated L-type calcium channels (Newgard and McGarry, 1995). In contrast, GLP-1 has been found to promote islet cell survival and proliferation by activation of the cAMP pathway (Hui et al., 2003). Transgenic mice expressing a dominant negative CREB polypeptide in islets develop diabetes with apoptosis of insulin producing beta cells due in part to reduced expression of IRS2, a direct target of CREB activity (Jhala et al., 2003).
cAMP promotes the expression of cellular genes by triggering the PKA mediated phosphorylation of CREB at Ser133 (Gonzalez and Montminy, 1989). Phosphorylation of CREB at Ser133 in turn stimulates target gene expression by enhancing recruitment of the histone acetylase coactivator paralogs CBP and P300 (Arias et al., 1994; Chrivia et al., 1993; Kwok et al., 1994). The structure of the CREB:CBP complex, using relevant interaction domains, called KID and KIX, respectively, reveals that phospho (Ser133) forms direct contacts with residues in KIX that account for half of the free energy of complex formation (Parker et al., 1998; Radhakrishnan et al., 1997). Binding of KID to KIX also promotes a random coil to helix transition in KID that favors formation of hydrophobic contacts with residues lining a shallow groove in KIX.
In addition to cAMP, CREB is Ser 33 phosphorylated in response to a number of stimuli, including growth factors, shear stress, and UV light (Mayr and Montminy, 2001). A number of these stimuli, however, are incapable of promoting target gene activation via CREB per se due in part to secondary phosphorylation of CREB at inhibitory sites. Neuronal depolarization triggers phosphorylation of CREB not only at Ser133 but also at Ser142 and Ser143 (Kornhauser et al., 2002), for example, and these modifications destabilize the CREB:CBP complex by electrostatic repulsion (Kornhauser et al., 2002; Parker et al., 1998).
The ability of calcium signals to promote CREB dependent transcription while apparently blocking CBP recruitment, at least via the KID domain, is indicative of the potential presence of other coactivators that either mitigate these effects or function independently of CBP/P300. The involvement of a distinct CREB coactivator in promoting calcium dependent gene expression is further indicated by studies in which addition of calcineurin antagonists are observed to block calcium-stimulated CREB activity without affecting levels of CREB Ser133 phosphorylation (Schwaninger et al., 1995). The identification of such putative coactivator(s), however, remains elusive.
Although the KID domain in CREB is thought to mediate target gene activation in response to most extracellular stimuli, other regions, most notably the bZIP DNA binding/dimerization domain, have also been implicated in this process. In previous studies using GAL4-CREB fusion proteins to define domain requirements for transcriptional activation, for example, both KID and bZIP domains were found to contribute importantly to cAMP and KCl responsiveness (Bonni et al., 1995a; Sheng et al., 1991). These results are also indicative of the involvement of additional cofactors that promote cAMP and calcium dependent transcription through an interaction with the CREB bZIP domain. Consistent with the ability of this region to recruit components of the transcriptional apparatus, the CREB bZIP domain has been found to act as a potent repressor of numerous transcription factors when over-expressed in various cells (Lemaigre et al., 1993).
Accordingly, there is a need in the art for methods to identify compounds that modulate the above-described interactions. Such compounds will find use in a variety of applications, such as, for example, enhancing islet cell activity and/or survival, promoting CREB-mediated gene expression in islet cells, effecting transport of Transducers of Regulated CREB (TORCs) from the cytoplasm into the nucleus of islet cells, effecting interactions between TORCs and member(s) of the 14-3-3 family of proteins, and the like.